Amyloid-β (Aβ)-containing extracellular plaques and hyperphosphorylated tau-loaded intracellular neurofibrillary tangles are neuropathological hallmarks of Alzheimer's disease (AD). Although Aβ exerts neuropathogenic activity through tau, the mechanistic link between Aβ and tau pathology remains unknown. Here, we showed that the FcγRIIb-SHIP2 axis is critical in Aβ1-42-induced tau pathology. Fcgr2b knockout or antagonistic FcγRIIb antibody inhibited Aβ1-42-induced tau hyperphosphorylation and rescued memory impairments in AD mouse models. FcγRIIb phosphorylation at Tyr273 was found in AD brains, in neuronal cells exposed to Aβ1-42, and recruited SHIP2 to form a protein complex. Consequently, treatment with Aβ1-42 increased PtdIns(3,4)P2 levels from PtdIns(3,4,5)P3 to mediate tau hyperphosphorylation. Further, we found that targeting SHIP2 expression by lentiviral siRNA in 3xTg-AD mice or pharmacological inhibition of SHIP2 potently rescued tau hyperphosphorylation and memory impairments. Thus, we concluded that the FcγRIIb-SHIP2 axis links Aβ neurotoxicity to tau pathology by dysregulating PtdIns(3,4)P2 metabolism, providing insight into therapeutic potential against AD.https://doi.org/10.7554/eLife.18691.001
In Alzheimer’s disease, damage to neurons in the brain gradually causes memory loss and difficulties with thinking. The main hallmarks of this damage are seen in the accumulation of proteins in and around neurons. First, a protein called amyloid beta forms aggregates outside the cell. This appears to lead to the build up of an abnormal form of a protein called tau inside the cell. These abnormal tau proteins have excessive numbers of phosphate groups attached to them, and so are known as “hyperphosphorylated”. The molecular mechanism underlying amyloid beta’s role in the hyperphosphorylation of tau proteins was not known.
Amyloid beta binds to many different receptor proteins – including one called Fc gamma receptor IIb – on the surface of neurons. Kam, Park et al. investigated whether interactions between amyloid beta and Fc gamma receptor IIb might regulate the phosphorylation of tau within neurons. Adding amyloid beta to mouse neurons caused tau proteins to become hyperphosphorylated. However, removing Fc gamma receptor IIb from the neurons, or stopping it from binding to amyloid beta, abolished this effect.
When amyloid beta was bound to Fc gamma receptor IIb, the receptor became phosphorylated. This in turn triggered a series of further phosphorylation events, culminating in an increase in the level of a molecule that relays signals from cell receptors – called SHIP2 – inside the neurons. This molecule increases tau phosphorylation when added to neurons. Reducing the activity or amount of SHIP2 in mice that present the symptoms of Alzheimer’s disease reduced the hyperphosphorylation of the tau protein in their neurons and restored their memory to normal levels.
Kam, Park et al. also looked at samples taken from the brains of human Alzheimer's disease patients. Unlike samples taken from people without Alzheimer’s disease, neurons in these samples contain both phosphorylated Fc gamma receptor IIb and hyperphosphorylated tau proteins.
By uncovering the molecules that link amyloid beta with tau hyperphosphorylation, Kam, Park et al.’s results suggest new targets for therapies to treat the symptoms of Alzheimer’s disease. More research is now needed to investigate whether this could lead to the design of new drugs.https://doi.org/10.7554/eLife.18691.002
Alzheimer's disease (AD) is characterized by the progressive loss of memory and the neuronal degeneration (Mattson, 2004). The pathological hallmarks of AD are the presence of senile plaques consisting of Aβ peptide and neurofibrillary tangles (NFT) formed by abnormally hyperphosphorylated tau (Walsh and Selkoe, 2004). Aβ species are generated from amyloid precursor protein (APP) by β- and γ-secretases, and accumulate extracellularly (Haass and Selkoe, 2007). In general, Aβ contributes to AD pathology by exhibiting toxicity in susceptible neurons, facilitating tau hyperphosphorylation, disrupting proteasome activity, and triggering synaptic dysfunction (LaFerla et al., 2007; Kam et al., 2014). Tau is a microtubule-binding protein but dissociates from the microtubules and accumulates in neurons as it becomes highly phosphorylated at multiple sites, resulting in the impairment of microtubule assembly and function (Ballatore et al., 2007).
Accumulating evidence strongly indicate that these two hallmarks are strongly interrelated in AD. In the amyloid cascade hypothesis, tau is believed to be one of the major downstream targets of Aβ to produce neurotoxicity (Hardy and Selkoe, 2002). Aβ accelerates neurodegeneration in neuronal cells but not in tau-deficient neurons (Rapoport et al., 2002). Moreover, tau depletion in mutant APP transgenic mice prevented Aβ pathologies, including learning and memory impairment (Roberson et al., 2007). The role of Aβ in tau pathology was also shown in 3xTg-AD mice expressing APP, presenilin, and tau transgenes in which Aβ immunization reduced not only Aβ accumulation but also tau pathology (Oddo et al., 2004). In addition, higher levels of NFT have been observed in APPswe/P301L transgenic mice (Lewis et al., 2001) and in 3xTg-AD mice (Oddo et al., 2003). More importantly, tau hyperphosphorylation is frequently found in AD brains (Grundke-Iqbal et al., 1989). Apparently, tau kinases, such as glycogen synthase kinase-3β (GSK-3β), are activated by Aβ for tau phosphorylation in vitro and in vivo (Hoshi et al., 2003; Ma et al., 2006; Terwel et al., 2008; Park et al., 2012). All of these findings indicate the presence of a pathologic signal pathway starting with extracellular Aβ and ending in the phosphorylation of intracellular tau. However, the mechanism connecting the two pathologic hallmarks of AD remains unknown.
Phosphoinositides, the phosphorylated derivatives of phosphatidylinositol (PtdIns), such as PtdIns(3,4,5)P3, PtdIns(4,5)P2, and PtdIns(3,4)P2, are known to play a major role in signal transduction upon cellular stimulation (Di Paolo and De Camilli, 2006). Among them, the biological roles of PtdIns(3,4,5)P3 and PtdIns(4,5)P2 have been relatively well characterized in cell survival, proliferation, and synaptic function via their binding proteins (Bunney and Katan, 2010; Khuong et al., 2013), but the function of PtdIns(3,4)P2 is largely unknown. Unlike PtdIns(4,5)P2, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are formed when cells respond to signals (Zhang and Majerus, 1998; Lemmon, 2008). SH2 domain-containing phosphatidylinositol 5′-phosphatase (SHIP) removes 5′ phosphate from PtdIns(3,4,5)P3 to produce PtdIns(3,4)P2 (Damen et al., 1996). Increasing evidence has revealed that phosphoinositide metabolism is dysregulated in AD; specifically, the level of PtdIns(4,5)P2 is decreased in human and mouse AD brains, and in the primary cortical neurons exposed to oligomeric Aβ (Stokes and Hawthorne, 1987; Jope et al., 1994; Berman et al., 2008), and recovery of PtdIns(4,5)P2 deficiency prevents AD-related cognitive deficits in mouse models (McIntire et al., 2012; Zhu et al., 2015). However, how phosphoinositide metabolism, including levels of PtdIns(3,4)P2, is regulated by Aβ during AD pathogenesis and the consequences of its dysregulation in AD needs to be resolved.
Until now, Aβ was reported to bind to many receptors, including alpha7 nicotinic acetylcholine receptors (α7 nAChR), NMDA receptor, receptors for advanced glycation end- products (RAGE), Aβ-binding alcohol dehydrogenase (ABAD), the Ephrin-type B2 receptor (EphB2), cellular prion protein (PrPc), and paired immunoglobulin-like receptor B (PirB) (Yan et al., 1996; Wang et al., 2000; Lustbader, 2004; Snyder et al., 2005; Laurén et al., 2009; Cissé et al., 2011a; Kim et al., 2013). Although these receptors were shown to be responsible for Aβ neurotoxicity, especially memory impairment in AD mice, their role as neuronal receptors in Aβ-induced tau pathologies was limitedly shown in α7 nAChR and NMDA receptor (reviewed in Stancu et al., 2014). Of particular note, while α7 nAChR was reported to mediate Aβ-induced tau phosphorylation, the finding was based on in vitro and ex vivo system (Wang et al., 2003). Furthermore, evidence showing a correlation of the proposed molecular mechanism with pathologic evidence was not much provided. In particular, the CAMKK2-AMPK at down-stream of NMDA receptor was recently proposed to mediate the synaptotoxic effects of Aβ oligomers through tau phosphorylation and this event is very likely caused by NMDA receptor-induced increase of intracellular calcium, not by direct interaction of NMDA receptor with Aβ (Mairet-Coello et al., 2013). Therefore, a neuronal receptor that is important in Aβ-induced tau pathology needs to be elucidated.
Recently, we showed that Fc gamma receptor IIb (FcγRIIb) is also expressed in neurons and directly interacts with Aβ1-42 to mediate Aβ neurotoxicity, synaptic dysfunction, and memory impairment in AD pathogenesis (Nimmerjahn and Ravetch, 2008; Kam et al., 2013). Here, we show that FcγRIIb is phosphorylated at tyrosine 273 by Aβ1-42 in neurons and in AD brains, and that this phosphorylation recruits SH2 domain-containing phosphatidylinositol 5′-phosphatase 2 (SHIP2, INPPL1) to increase PtdIns(3,4)P2 levels for tau hyperphosphorylation. Further, Fcgr2b or Inppl1 deficiency in 3xTg-AD mice or pharmacological inhibition of either protein abrogates all of these observations, highlighting the importance of the FcγRIIb-SHIP2 axis in the Aβ-induced tau pathology.
Given that FcγRIIb was previously identified as a receptor for Aβ1-42 and a mediator of memory impairment in hAPP-J20 mice expressing only familial mutant APP (Kam et al., 2013), and that tau as well as Aβ is essential for memory impairment in AD mouse (Ramsden et al., 2005), we hypothesized that FcγRIIb is responsible for tau hyperphosphorylation in response to Aβ1-42. We first assessed and confirmed that both FcγRIIb mRNAs and proteins were significantly expressed in neurons as well as non-neuronal cells in mouse brains (Figure 1—figure supplement 1). Interestingly, incubation of primary cortical neurons with synthetic Aβ1-42 oligomers increased tau phosphorylation at several pathological epitopes, including Ser396/Ser404 (PHF1), Thr231/Ser235 (AT180), and Ser202 (CP13), but these phosphorylations were abrogated in Fcgr2b knockout (KO) neurons (Figure 1A,B). Immunocytochemical analysis also showed that immunoreactivity against the phosphorylated tau was increased by treatment with Aβ1-42 in the neuron-specific enolase (NSE)-positive cortical neurons, but not in the Fcgr2b KO neurons (Figure 1C).
Instead of using a high dose of synthetic Aβ1-42, we further utilized a low-dose of cell-derived, naturally secreted Aβ oligomers (Walsh et al., 2002). The presence of the secreted, soluble Aβ oligomers in the conditioned medium (CM) of 7PA2 cells was confirmed by western blot analysis, and its production was inhibited by treatment with a γ-secretase inhibitor, compound E (Figure 1—figure supplement 2A). We found that tau phosphorylation was increased in the primary hippocampal neurons that were cocultured with 7PA2 cells (Figure 1D), but not in the neurons cocultured with compound E-treated cells (Figure 1—figure supplement 2B). Further, we found that there was no hyperphosphorylation of tau in the Fcgr2b KO neurons that were cocultured with 7PA2 cells (Figure 1D). These results suggest that tau hyperphosphorylation in the cultured neurons was induced by physiologically relevant levels of Aβ oligomers through FcγRIIb.
The 3xTg-AD mice develop age-dependent and progressive Aβ and tau pathologies, including memory impairment (Oddo et al., 2003). Thus, in order to uncover the role of FcγRIIb in tau pathology, we crossed 3xTg-AD mice with Fcgr2b KO mice to generate double transgenic mice (3xTg-AD/Fcgr2b KO). Each group of mice was tested for spatial working memory in a Y-maze task. Although the total number of arm entries was not significantly different between groups, Fcgr2b deficiency improved the spatial working memory of 3xTg-AD mice (Figure 1E). In the novel object recognition test, only 3xTg-AD/Fcgr2b KO mice, but not 3xTg-AD mice, discriminated between novel and familiar objects in the second and third trials (Figure 1F), indicating that the recognition memory deficit of 3xTg-AD mice was prevented by Fcgr2b deficiency. Further, 3xTg-AD/Fcgr2b KO mice performed well in the passive avoidance task, whereas 3xTg-AD mice showed a deficit in passive avoidance memory (Figure 1G). These data indicate that FcγRIIb is required for the learning and memory impairments in 3xTg-AD mice.
Because FcγRIIb itself did not affect Aβ levels in these mice (Figure 1—figure supplement 3A) and the hippocampus of 3xTg-AD mice at nine months of age was plaque-free (Hirata-Fukae et al., 2008), we next examined the role of FcγRIIb in tau pathology in the mouse brains. As reported (Hirata-Fukae et al., 2008), the pathologic hyperphosphorylation of tau detected by CP13, PHF1, and AT180 antibodies was found to be increased in the 3xTg-AD mice showing memory impairment (Figure 1H,I). In contrast, genetic deletion of Fcgr2b in 3xTg-AD mice abolished the hyperphosphorylation of tau. In the brains of 20 month-old 3xTg-AD mice harboring Aβ plaques, tau phosphorylation was markedly elevated as compared to 6 month-old mice (Figure 1—figure supplement 3B,C). FcγRIIb deficiency also prevented the hyperphosphorylation of tau in 15 month-old 3xTg-AD mice (Figure 1—figure supplement 3D,E), indicating that tau phosphorylation mediated by FcγRIIb occurs at the onset of the disease and lasts to the late stage. Similarly, this inhibitory effect of Fcgr2b deficiency on tau phosphorylation was observed in another AD model, hAPP-J20 mice (Figure 1—figure supplement 3F,G). These results indicate that FcγRIIb is crucial for tau hyperphosphorylation in AD model mice showing memory impairment.
Because Aβ transduces toxic signals into the neurons via direct interaction with FcγRIIb (Kam et al., 2013), we examined whether this interaction is necessary for tau hyperphosphorylation. Compared to Aβ only-treated neurons, addition of the purified hFcγRIIb ectodomain (hFcγRIIb-ED) to culture medium blocked Aβ-induced tau hyperphosphorylation in primary cortical neurons (Figure 2A,B). With the notion that both Aβ1-42 oligomers and immunoglobulin complexes share the same binding site on FcγRIIb (Kam et al., 2013), we found that the incubation of cultured cells with anti-FcγRIIb antibody (2.4G2) blocked the interaction between Aβ1-42 and HA-tagged FcγRIIb (Figure 2—figure supplement 1A,B). This antibody selectively recognized FcγRIIb but did not crossreact with Aβ1-40 or Aβ1-42 (Figure 2—figure supplement 1C). Consistently, treatment with 2.4G2 antibody drastically decreased Aβ-induced tau hyperphosphorylation in mouse primary cortical neurons in a dose-dependent manner (Figure 2C,D). These observations suggest that the interaction between Aβ1-42 and FcγRIIb is an initial step for tau phosphorylation in the cultured neurons.
Next, we addressed the effects of 2.4G2 antibody on Aβ-induced acute memory impairment in mice. When Aβ1-42 was directly injected into the intracerebroventricular (i.c.v.) region of wild-type (WT) mice (Kam et al., 2013), the mice showed impaired behaviors (Figure 2E–G). Interestingly, coinjection of Aβ1-42 and 2.4G2 antibodies significantly rescued the deficits of spontaneous alternation behavior (Figure 2E), object recognition memory (Figure 2F), and passive avoidance memory (Figure 2G). On the other hand, injection with normal immunoglobulin G (IgG) did not exhibit such inhibitory effects on memory impairment. There was no difference in total movements between these groups of mice, as reflected by total arm entry in the Y-maze test (Figure 2—figure supplement 1D). From western blot analysis, we found that tau phosphorylation was highly increased in the hippocampus of Aβ1-42-injected mice (Figure 2H). In contrast, Aβ-induced tau phosphorylation was abolished in the hippocampus by coinjection with 2.4G2 antibody, but not with IgG (Figure 2H). Thus, we found that the interaction between Aβ1-42 and FcγRIIb is essential for tau hyperphosphorylation and memory impairment in mice.
As reported (Plattner et al., 2006), we found that tau kinases, such as GSK3β and Cdk5, were activated for tau phosphorylation in cultured neurons by treatment with Aβ1-42 (Figure 3—figure supplement 1A,B). Notably, Fcgr2b deficiency in primary cortical neurons abrogated the activation of GSK3β and tau phosphorylation triggered by Aβ1-42, but not that of Cdk5. Conversely, ectopic expression of FcγRIIb induced tau hyperphosphorylation, detected by PHF1, CP13, and AT180 antibodies, in both SH-SY5Y cells and primary cortical neurons (Figure 3—figure supplement 1C–F). Consistently, treatment with SB-415286, a GSK3β inhibitor, markedly prevented FcγRIIb-induced tau hyperphosphorylation at those epitopes (Figure 3—figure supplement 1C,E), whereas roscovitine, a Cdk5 inhibitor, did not affect tau phosphorylation (Figure 3—figure supplement 1D,F). These observations suggest that FcγRIIb transduces Aβ signal into the neuronal cells to activate GSK3β for tau hyperphosphorylation.
We next investigated the mechanism for FcγRIIb transduction of Aβ signal into the neurons for tau phosphorylation, and found that treatment with Aβ1-42 induced phosphorylation at Tyr273 within an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytosolic region of FcγRIIb in SH-SY5Y cells (Figure 3A). This phosphorylation of FcγRIIb was also detected in Aβ-treated primary cortical neurons, but not in Fcgr2b KO neurons (Figure 3B). Further, addition of purified hFcγRIIb-ED protein prevented Aβ-induced phosphorylation of FcγRIIb-Tyr273 in SH-SY5Y cells (Figure 3C), indicating that FcγRIIb is phosphorylated on Tyr273 after its interaction with Aβ1-42.
By using FcγRIIb mutants, we further characterized the role of FcγRIIb phosphorylation in neuronal cells. We generated several FcγRIIb mutants lacking the ITIM (ΔITIM) or cytoplasmic region (Δcyto), or substituting the tyrosine residue with phenylalanine (Y273F), and examined their effects on Aβ1-42 neurotoxicity and tau phosphorylation. Unlike FcγRIIb, ectopic expression of FcγRIIb mutants did not induce neuronal death in mouse hippocampal HT22 cells (Figure 3—figure supplement 2A). Moreover, overexpression of FcγRIIb Y273F mutant, which was not phosphorylated (Figure 3D), blocked Aβ1-42 neurotoxicity to the control level in SH-SY5Y cells, whereas FcγRIIb WT potentiated it (Figure 3—figure supplement 2B), indicating that FcγRIIb-Tyr273 phosphorylation is required for Aβ1-42 neurotoxicity. Next, we examined the role of FcγRIIb phosphorylation in tau phosphorylation using a reconstitution analysis in Fcgr2b KO neurons. Unlike that in Fcgr2b-deficient neurons, reconstitution of the Fcgr2b KO primary cortical neurons with FcγRIIb WT recovered Aβ-induced hyperphosphorylation of tau, detected by PHF1, CP13, AT180, and 12E8 antibodies, as seen in WT neurons (Figure 3E). On the other hand, reconstitution with FcγRIIb-ΔCyto or FcγRIIb-Y273F failed to show tau phosphorylation at those epitopes in response to Aβ1-42 (Figure 3E). These results suggest that the phosphorylation of FcγRIIb-Tyr273 by Aβ1-42 is critical for tau phosphorylation.
Even more interestingly, when we analyzed the phosphorylation status of FcγRIIb in the brains of AD patients, we found FcγRIIb phosphorylation on Tyr273 in the hippocampal tissues of five out of six AD patients (stage V and VI), but not in normal and mild cognitive impairment (MCI) patients (stage III) (Figure 3F). In addition, tau phosphorylation at PHF was observed in four out of the five AD patients with FcγRIIb-Tyr273 phosphorylation-positive brains. As we previously reported, the expression level of FcγRIIb was increased in AD brains (Figure 3F). With the notion that FcγRIIb phosphorylation is required for tau phosphorylation and Aβ1-42 neurotoxicity, it is likely that the phosphorylation of FcγRIIb found in AD brains is associated with tau phosphorylation and neuronal loss during AD pathogenesis.
In B cells, the SHIP (SHIP1 and 2) is known to bind to the phosphorylated ITIM region of FcγRIIb and inhibit downstream responses triggered by immune receptors (Ono et al., 1996; Muraille, 2000). Given that SHIP2 is highly expressed in the brain and SHIP1 is expressed predominantly in hematopoietic cells (Astle et al., 2007), we focused on SHIP2 and examined its ability to bind to phosphorylated FcγRIIb in neuronal cells. Immunoprecipitation assays revealed that overexpressed FcγRIIb-GFP bound to SHIP2-His in SH-SY5Y cells, whereas the phospho-defective FcγRIIb (Y273F) mutant failed to do so (Figure 4A). Similar results were observed in the reverse immunoprecipitation assay using GFP antibody (Figure 4B). Interestingly, compared to the lack of or weak interaction in untreated control cells, we found a drastic increase in the binding between endogenous SHIP2 and FcγRIIb in SH-SY5Y cells after exposure to Aβ1-42 (Figure 4C,D), suggesting that SHIP2 binds to FcγRIIb in neuronal cells in response to Aβ1-42.
Because SHIP2 is known to be localized in the cytosol but to translocate to the cell membrane upon stimulation (Dyson et al., 2001; Wang et al., 2004), we further examined subcellular localization of SHIP2 in neuronal cells exposed to Aβ1-42. Treatment of SH-SY5Y cells with Aβ1-42 enhanced subcellular localization of SHIP2 to the plasma membrane and thus colocalization of SHIP2 with FcγRIIb (Figure 4E). In addition, subcellular fractionation assays revealed similar results, showing that SHIP2 was enriched in the membrane fraction following Aβ1-42 treatment, while it was detected in both the cytosol and membrane fractions in untreated control cells (Figure 4F). However, SHIP2 expression was not changed by Aβ1-42 treatment in SH-SY5Y cells and in the brains of MCI and AD patients (Figure 4F andFigure 4—figure supplement 1). Overall, our data indicates that SHIP2 is recruited to the membrane to interact with FcγRIIb in neuronal cells following Aβ1-42 treatment.
Because SHIP2 is an inositol phosphatase that dephosphorylates PtdIns(3,4,5)P3 to produce PtdIns(3,4)P2 (Damen et al., 1996), we examined whether Aβ1-42 affects phosphoinositide metabolism through FcγRIIb and SHIP2. We measured the levels of PtdIns(3,4)P2 with a protein lipid overlay (PLO) assay using the purified His-tagged pleckstrin homology (PH) domain of tandem PH domain-containing protein-1 (TAPP1) as a probe (Dowler et al., 2002). From the PLO assays using total lipid extracts, we found that the amount of TAPP1-PH-interacting PtdIns(3,4)P2 was increased in SH-SY5Y cells following exposure to Aβ1-42 (Figure 5—figure supplement 1A). In contrast, there was no such increase by Aβ1-42 in SH-SY5Y/FCGR2B or SH-SY5Y/INPPL1 knockdown cells (Figure 5—figure supplement 1B). Conversely, the amount of the general receptor for phosphoinositides-1 (GRP1)-PH-interacting lipids, mainly PtdIns(3,4,5)P3 (Guillou et al., 2007), was reduced by Aβ1-42 in SH-SY5Y cells, but not in SH-SY5Y/FCGR2B or SH-SY5Y/INPPL1 knockdown cells (Figure 5—figure supplement 1B). Similar patterns of changes in the levels of TAPP1-PH-interacting or GRP1-PH-interacting lipids were also observed in WT primary cortical neurons after treatment with Aβ1-42 (Figure 5A,B). Consistently, these changes were not observed in Fcgr2b KO neurons. These results indicate that phosphoinositide metabolism is affected by the FcγRIIb-SHIP2 axis.
When we directly measured the levels of phosphoinositides with an ELISA assay, we observed that the levels of PtdIns(3,4)P2 were increased by 30% in primary cortical neurons after exposure to Aβ1-42, whereas PtdIns(3,4,5)P3 was decreased by 17% (Figure 5C). However, Fcgr2b-deficiency abrogated these changes in PtdIns(3,4)P2 and PtdIns(3,4,5)P2 levels. On the other hand, the levels of PtdIns(4,5)P2 were lowered by Aβ1-42 in WT neurons, consistent with a previous report (Berman et al., 2008), and also in FcgR2b KO neurons (Figure 5C). We further traced the changes in PtdIns(3,4)P2 using GFP-tagged TAPP1-PH under a fluorescence microscope. While TAPP1-PH-GFP was found in a diffuse pattern in the cytosol of untreated SH-SY5Y cells, treatment with Aβ1-42 enhanced the fluorescence of TAPP1-PH-GFP and concentrated it at the plasma membrane (Figure 5D). In contrast, Aβ-induced accumulation of TAPP1-PH-GFP at the plasma membrane was impaired in SH-SY5Y/FCGR2B knockdown cells (Figure 5D). As reported (Cheung et al., 2007), hydrogen peroxide also induced membrane localization of TAPP1-PH-GFP in SH-SY5Y cells. However, this localization was not affected by Fcgr2b deficiency (Figure 5—figure supplement 1C). These observations further support the idea that Aβ1-42 selectively dysregulates PtdIns(3,4)P2 and PtdIns(3,4,5)P3 levels in neurons via FcγRIIb.
To address the important question of whether the increase in PtdIns(3,4)P2 by Aβ1-42 can influence tau phosphorylation, we directly delivered phosphoinositide into living neurons using a carrier (Ozaki et al., 2000). Compared to untreated control cells, treatment with PtdIns(3,4)P2 increased tau phosphorylation (AT180, CP13, PHF1) in primary cortical neurons in a dose-dependent manner (Figure 5E). Interestingly, the increase in tau hyperphosphorylation was specific to PtdIns(3,4)P2; other phosphoinositides, such as PtdIns(4,5)P2, PtdIns(3,5)P2, and PtdIns(3,4,5)P3, failed to do so. Consistent with the activation of GSK3β by Aβ1-42, PtdIns(3,4)P2 treatment also reduced the inhibitory phosphorylation of GSK3β at Ser9 in neurons (Figure 5—figure supplement 2A). Moreover, PtdIns(3,4)P2 induced the expression of GRP78, a typical marker of unfolded protein response (UPR), as well (Figure 5—figure supplement 2A). We further found that PtdIns(3,4)P2-induced GSK3β activation and tau phosphorylation were attenuated by the treatment with ER stress inhibitors, such as 4-PBA and Salubrinal, a chemical chaperone and an eIF2α dephosphorylation inhibitor, respectively (Figure 5—figure supplement 2B). We confirmed that ER stress response and GSK3β activation triggered by Aβ were all declined by a SHIP2 inhibitor AS1949490 or lentiviral expression of Inppl1 siRNA (Figure 5—figure supplement 2C,D). Combined with the previous study showing that ER stress stimulates GSK3β activity (Ren et al., 2015), these results suggest that an increase in the PtdIns(3,4)P2 level by Aβ1-42 activates GSK3β through ER stress for tau hyperphosphorylation in neuronal cells.
We further determined whether SHIP2, a downstream signal mediator of FcγRIIb, is essential for tau phosphorylation by examining the effects of SHIP2 knockdown. Unlike enhanced tau phosphorylation by Aβ1-42 in control cells, knockdown of SHIP2 expression in SH-SY5Y cells abrogated tau phosphorylation by Aβ1-42 (Figure 6—figure supplement 1A). Conversely, overexpression of SHIP2 alone was sufficient to increase tau hyperphosphorylation in SH-SY5Y cells (Figure 6—figure supplement 1D). On the other hand, these effects were not observed using an activity-dead SHIP2 mutant with Asp608 replaced by Ala (Nakatsu et al., 2010) (Figure 6—figure supplement 1E). In addition, neuronal cell death triggered by Aβ1-42 treatment or FcγRIIb overexpression was greatly reduced by knockdown of SHIP2 expression in SH-SY5Y cells (Figure 6—figure supplement 1B,C). Together, these data suggest that SHIP2 is a key signal mediator of FcγRIIb in Aβ-induced tau hyperphosphorylation and neurotoxicity.
Then, we examined the role of SHIP2 (INPPL1) in memory impairment in vivo using Inppl1 siRNA-expressing lentivirus (lenti-siInppl1). We confirmed that infection with lenti-siInppl1 reduced SHIP2 levels in both HT22 cells and primary cortical neurons (Figure 6—figure supplement 2A,B). Consistently, we observed that infection with lenti-siInppl1 abrogated Aβ-induced tau phosphorylation (PHF1, CP13) in primary cortical neurons (Figure 6A). When we stereotaxically injected lenti-siInppl1 into the dentate gyrus of WT and 3xTg-AD mice, we observed that compared to control 3xTg AD mice, lenti-siInppl1-injected 3xTg AD mice showed no significant memory deficits in Y-maze and novel object recognition tests at 20 days after viral injection (Figure 6B,C). When we also monitored tau phosphorylation and SHIP2 levels in brains by western blot analysis, we found that tau phosphorylation and SHIP2 levels were reduced in the hippocampi of lenti-siInppl1-injected 3xTg-AD mice (Figure 6D). The amelioration of memory impairment by lenti-siInppl1 was maintained for 30 days post-injection (data not shown). These results indicate that SHIP2 is critical to memory impairment and tau hyperphosphorylation in 3xTg-AD mice.
In addition, we assessed the impact of pharmacological SHIP2 inhibition on Aβ-induced tau phosphorylation and memory impairment using a SHIP2-selective inhibitor, AS1949490, which is a 30-fold more potent inhibitor against SHIP2 than SHIP1 (Suwa et al., 2009). Treatment of primary cortical neurons with AS1949490 inhibited Aβ-induced tau hyperphosphorylation and neuronal cell death (Figure 6E and Figure 6—figure supplement 3A). When we examined the effect of AS1949490 on the memory impairment triggered by i.c.v.-injected Aβ1-42 in mice, we directly delivered AS1949490 into mouse brains because of its poor bioavailability (Suwa et al., 2010). Behavioral tests following the i.c.v. injection of sub-lethal doses of Aβ1-42 and AS1949490 revealed that AS1949490 significantly rescued Aβ1-42-induced impairments of spatial working memory (Figure 6F), object recognition memory (Figure 6G), and passive avoidance memory (Figure 6H). Total movements, determined by arm entries in the Y-maze test, were not significantly different between the groups of mice (Figure 6—figure supplement 3B). Moreover, from western blot analysis of the mouse hippocampal tissues, we found that AS1949490 suppressed Aβ1-42-induced tau phosphorylation (PHF1, CP13, AT180) (Figure 6I). Taken together, these observations support the view that pharmacological manipulation of SHIP2 is amenable to the development of AD therapeutics targeting Aβ1-42-induced tau phosphorylation and memory impairment.
Despite tremendous efforts showing that Aβ plays a central role in the pathogenesis of AD, including memory impairment, synaptic loss, and neuronal cell death (Cleary et al., 2005; LaFerla et al., 2007), mechanistic understanding of tau phosphorylation in Aβ-induced memory deficits remains poor (Rapoport et al., 2002; Roberson et al., 2007; Shipton et al., 2011). To our knowledge, this is the first study showing crucial mediator, the FcγRIIb-SHIP2 axis, which is responsible for Aβ-induced tau phosphorylation, memory impairment, and neuronal loss in AD models. Because the interaction of Aβ species, especially oligomeric Aβ1-42, with FcγRIIb accounts for such neuropathogenic defects of AD as an initiation step, the selective interaction of oligomeric Aβ1-42 with FcγRIIb may hint at why Aβ1-42, but not Aβ1-40, is important in tau pathology (Oddo et al., 2008; Kam et al., 2013). Consequently, inhibition of the interaction between FcγRIIb and Aβ using anti-FcγRIIb antibody prevents Aβ-induced tau phosphorylation and memory deficits.
In general, Aβ oligomers play a key role in AD pathogenesis, while soluble Aβ oligomers are still heterogeneous, including low or high n oligomers, and the proposals on which species of Aβ oligomers are responsible for the pathogenesis are a little in debate (reviewed in Benilova et al., 2012). We have here used 3 different sources of Aβ oligomers; synthetic Aβ oligomers, naturally secreted Aβ oligomers (7PA2 cells), and Aβ of 3xTg-AD model mice. Although synthetic Aβ oligomers are well-characterized and have been used widely for neurotoxicity, the acting concentration of synthetic Aβ (μM range) is relatively higher than that in AD brains. Compared to synthetic Aβ oligomers, conditioned medium from 7PA2 cells mainly contains not only Aβ dimers and trimers but also the different pools of Aβ oligomers, including zeta peptide, and show more potent neurotoxic properties (nM range) (Qi-Takahara, 2005; Haass and Selkoe, 2007). Moreover, the oligomers generated in 3xTg-AD mouse brain may be more complicated and needs to be identified. Nonetheless, we propose here that FcγRIIb plays a crucial role in tau phosphorylation and neurotoxicity in vitro and in vivo in response to these species of Aβ, probably a certain common species among the different sources of Aβ oligomers.
Given that FcγRIIb was initially reported as a hematopoietic receptor which is mainly expressed in B cells, macrophages, and neutrophils (Nimmerjahn and Ravetch, 2008), our data using 3xTg-AD/FcγRIIb KO mice raised a possibility that FcγRIIb in non-neuronal cells might contribute to APP/Aβ-induced tau pathologies via neuroinflammatory function. As expected, we observed that microglia was activated in 3xTg-AD mice and this activation was also reduced significantly in the cortex and marginally in the hippocampus by FcγRIIb deficiency (data not shown). Recently, however, we and other colleagues identified that both FcγRIIb mRNA and protein are also expressed in neurons (Figure 1—figure supplement 1; Cahoy et al., 2008; Suemitsu et al., 2010; Kam et al., 2013), though FcγRIIb expression in neurons is low compared to astrocytes and immune tissues. Notably, its expression is increased at least several folds in primary neurons after exposure to Aβ1-42 and in AD brain (Kam et al., 2013). In addition, transgenic expression of the FcγRIIb mutant lacking its cytoplasmic domain in the neurons blocked memory impairment in 3xTg AD mice (data not shown). Moreover, the results showing that Aβ-induced tau phosphorylation was prevented by FcγRIIb deficiency, antagonistic FcγRIIb antibody, or SHIP2 inhibitor in primary cultured neurons assure neuronal function of the FcγRIIb-SHIP2 axis. Together, we believe that the inflammation mediated by FcγRIIb in 3xTg-AD mice is not likely a major cause of the memory impairment but contributes to the aggravation of memory impairment in the mice.
Unlike other FcγRs that have an immunoreceptor tyrosine-based activating motif (ITAM), FcγRIIb has a unique ITIM in its cytoplasmic tail and thus acts as an inhibitory receptor in B cells (Okun et al., 2010). Excitingly, the binding of extracellular Aβ1-42 to FcγRIIb induces phosphorylation in the ITIM of FcγRIIb in neuronal cells. We observed that Lyn kinase is expressed in neurons and that knockdown of LYN expression blocks Aβ-induced FcγRIIb phosphorylation and neurotoxicity (data not shown). Thus, it is likely that Lyn phosphorylates the ITIM of FcγRIIb in neuronal cells in response to Aβ1-42. Moreover, we also observed this phosphorylation of FcγRIIb in AD brains (stage V and VI) in which tau was highly phosphorylated. Then, how is FcγRIIb different from other Aβ receptors? It is reasonable to propose that different mechanisms or even the same mechanism exerts multiple effects at different stages of disease progression (De Strooper and Karran, 2016). For instance, RAGE is now believed to mainly function to transport Aβ in the blood brain barrier (Deane et al., 2003) and ABAD acts for mitochondrial toxicity as an intracellular binding partner of Aβ (Lustbader, 2004). In case of PrPc, it's debatable whether it is involved in Aβ-induced memory impairments and thus needs to be further characterized (Balducci et al., 2010; Gimbel et al., 2010; Cissé et al., 2011b). Further, compared to those receptors, our observations that the phosphorylation of FcγRIIb at tyrosine 273 is found in the brain of AD patients and is required for both oligomeric Aβ neurotoxicity and tau hyperphosphorylation can make it distinct from other Aβ-binding receptors. In the case of PirB that shares structural similarity with FcγRIIb and also acts as an Aβ receptor for synaptic plasticity, the phosphorylation of PirB is not associated with Aβ signaling (Kim et al., 2013). Thus, we believe that FcγRIIb facilitates tau phosphorylation and neuronal loss in AD brains, consistent with the proposed role of tau in AD pathogenesis, such as severe memory impairment and neuronal loss (Ballatore et al., 2007).
Interestingly, we show that SHIP2 is a key mediator in delivering the toxic signal of Aβ1-42 to tau by binding to the phosphorylated FcγRIIb. Many studies on SHIP2 have focused on its inhibitory effect on insulin signaling (Ishihara et al., 1999; Wada et al., 2001). Inppl1 transgenic mice show impaired insulin signaling and glucose intolerance, while Inppl1 KO mice are highly resistant to weight gain on a high-fat diet (Sleeman et al., 2005; Kagawa et al., 2008). Thus, targeting SHIP2 is thought to be a promising approach for the treatment of other diseases, including type 2 diabetes (Vanhanen et al., 2006). Given that metabolic syndrome, including insulin resistance and glucose intolerance, is highly associated with AD (Vanhanen et al., 2006; Razay et al., 2007), we speculate that SHIP2 may play a dual role in AD and diabetes. In support of this, a SHIP2 inhibitor exhibited an ameliorating effect on the impaired memory function of diabetic mice (Soeda et al., 2010). Several single-nucleotide polymorphisms (SNPs) of SHIP2 are involved in metabolic syndrome (Kaisaki et al., 2004; Kagawa et al., 2005); thus, it will be very interesting to evaluate the effect of these SHIP2 SNPs on AD pathogenesis in our FcγRIIb-SHIP2 axis.
Dysregulation of phosphoinositide metabolism is increasingly recognized as important in various diseases, such as cancer, diabetes, and myopathy (Wymann and Schneiter, 2008; Kok et al., 2009). The phosphoinositide pool is also altered in AD (Stokes and Hawthorne, 1987; Jope et al., 1994). In particular, this work shows that a change in the PtdIns(3,4)P2 level is implicated in AD pathogenesis through tau hyperphosphorylation. PtdIns(3,4)P2 is scarce under normal conditions and increases through signaling (Lemmon, 2008). While PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are known to overlap to some degree in their functions, they apparently have distinct roles in neurodegeneration. Recently, it was shown that PtdIns(3,4)P2, but not PtdIns(3,4,5)P3 and PtdIns(4,5)P2, potentiates glutamate-induced cell death in neurons (Sasaki et al., 2010). In addition, PtdIns(3,4)P2 phosphatase INPP4A deficiency shows increased level of PtdIns(3,4)P2 and leads to neurodegeneration in brains (Sasaki et al., 2010). We also observed that PtdIns(3,4)P2 selectively induces tau hyperphosphorylation in neurons. In addition, tensin homolog deletion on chromosome 10 (PTEN) also dephosphorylates PtdIns(3,4,5)P3 at position 3 and generates PtdIns(4,5)P2 (Li et al., 1997). The reduced level of PTEN in AD brains correlates with an increase in tau phosphorylation and, thus, dysregulation of PTEN also contributes to tau pathology (Kerr et al., 2006; Zhang et al., 2006). More recently, genetic reduction of synaptojanin 1 (Synj1), the major PtdIns(4,5)P2 phosphatase in the brain, ameliorates behavioral and synaptic deficits and accelerates Aβ clearance through rescuing PtdIns(4,5)P2 deficiency (McIntire et al., 2012; Zhu et al., 2015). Because oligomeric Aβ decreases PtdIns(4,5)P2 levels in AD (Berman et al., 2008), as we also observed, there is a possibility that SHIP2 activation together with either Synj1 upregulation or PTEN downregulation induces the imbalance in the phosphoinositide pool between PtdIns(3,4)P2 and PtdIns(4,5)P2, and leads to AD pathogenesis.
In conclusion, the FcγRIIb-SHIP2 signaling axis provides the missing link between Aβ and tau pathologies. Notably, Aβ1-42 induces FcγRIIb phosphorylation to recruit SHIP2, leading to disruption of phosphoinositide metabolism for tau hyperphosphorylation and memory impairment in neurons and AD model mice. Together with the Aβ-lowering strategy, our results provide new ways for AD therapeutics to rescue Aβ and tau pathology: i) selective inhibition of the interaction between FcγRIIb and Aβ1-42, ii) inhibition of a kinase (i.e., Lyn) which phosphorylates FcγRIIb upon Aβ1-42 stimulation, and iii) inhibition of SHIP2 which disrupts phosphoinositide metabolism.
WT (C57BL/6), Fcgr2b KO C57BL/6 (Takai et al., 1996), 3xTg-AD and hAPP (J20, The Jackson Laboratory, Bar Harbor, ME) mice were used. All experiments involving animals were performed according to the protocols approved by the Seoul National University Institutional Animal Care and Use Committee (SNU IACUC) guidelines. For biochemical assays, mice were anesthetized and the brains were rapidly dissected into subregions (cortex and hippocampus) and snap-frozen at –80°C. The hemibrains were homogenized in lysis buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, protease inhibitor cocktail] and centrifuged at 14,000 g for 20 min at 4°C. Supernatant was then collected and the protein concentration was determined using the Bradford method (GE Healthcare).
Hippocampal tissues from AD (Braak V-VI) (mean age, 80.2 ± 10.6 years; mean post-mortem intervals, 16.3 ± 6.7 hr), MCI (Braak III) (mean age, 84.2 ± 2.9 years; mean post-mortem intervals, 20.1 ± 8.0 hr) and non-AD patients (mean age, 67.8 ± 16.5 years; mean post-mortem intervals, 20.1 ± 5.8 hr) were kindly provided by the Harvard Brain Tissue Resource Center (McLean Hospital). Hippocampal tissues of AD patients were homogenized in ice-cold Tris-buffered saline (TBS) buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl and protease inhibitor cocktails]. The homogenates were clarified by centrifugation at 14,000 g for 20 min at 4°C, aliquoted and stored at –80°C until use. The supernatants were subjected to SDS-PAGE.
Seven to 8-month-old WT and 3xTg-AD mice were deeply anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). Lentivirus expressing shRNA against mouse Inppl1 (5′-GAA GGG AGG GCA CGT TAA TTT-3′) (Sigma-Aldrich) was used for injection and pLKO.1-Neo-CMV-tGFP non-target virus was used as a control. The lentivirus (1.1 × 1099 TU/ml, TU; transduction unit) was stereotaxically injected bilaterally into the dentate gyrus (2 μl per hemisphere at 0.4 μl/min) with the following coordinates: anteroposterior = 2.1 mm from bregma, mediolateral = ± 1.8 mm, dorsoventral = 2.0 mm. After the injection, the cannula was maintained for an additional 5 min for a complete absorption of the virus. Behavior tests were performed 20 and 30 days after the injection. For western blot analysis, brains were removed 25 days after viral injection, hippocampal region was dissected and its protein samples were prepared as described above. Intracerebroventricular injection of PBS or Aβ1-42 (Sigma-Aldrich, St. Louis, MO) was performed as described previously (Kam et al., 2013). In the case of coinjection experiments, oligomeric Aβ1-42 was incubated alone or together with AS1949490, IgG or 2.4G2 antibody before use.
Behavior tests for double transgenic or injected mice were performed as described previously (Kam et al., 2013). All apparatus and objects were cleaned with 70% ethanol before and after each trial. In Y-maze test, the mice were placed in the end of one arm (32.5 cm length × 15 cm height) of apparatus and allowed to move freely for 7 min. When the all four paws were into the arm, the entry was counted. The percentage of spontaneous alternations was calculated as the ratio of the number of successful alternations to the number of total alternations. In novel object recognition test, the mice were habituated in a chamber (22 cm wide × 27 cm long × 30 cm high) for 7 min with 24 hr intervals. In training trial #1 (2 days after habituation), the mice were exposed to two objects and allowed to explore freely for 7 min. In the testing trial #2 (a day after training), one of the familiar objects was replaced to a novel object and recognition was counted for 7 min. The same test was repeated after 24 hr, but with another novel object (trial #3). The object recognition was defined as spending time with orienting toward the object in a distance of 1 cm or less, sniffing the object or touching with the nose. The passive avoidance test was done in an apparatus consist of a light and dark compartment (20 × 20 × 20 cm each) separated by a guillotine door. The mice were allowed to explore the box for 5 min with the door open for habituation, and then returned to home cage. After 24 hr, the mice were placed into the light compartment for conditioning. The door was closed after entering into the dark room and then an electric foot shock (0.25 mA, 2 s) was delivered by the floor grids. The latency time for mice to enter the dark room was measured with a 5 min cut-off after 24 hr.
Synthetic Aβ1-42 oligomers were prepared from lyophilized monomers (rPeptide, Bogart, GA). The hydroxyfluroisopropanol (HFIP)-treated Aβ1-42 peptide was dissolved in dimethylsulfoxide (DMSO) and then diluted in phosphate-buffered saline (PBS). The stock solution was incubated at 4°C for 24 hr and stored at –80°C until use. Before use, the solution was centrifuged at 12,000 g for 10 min and the supernatant was used as an oligomeric Aβ (ADDLs). The oligomeric status of Aβ1-42 was evaluated by western blot analysis and atomic force microscopy (Kam et al., 2013). The conditioned medium of 7PA2-CHO cells (kindly provided by Dr. D.J. Selkoe, Harvard Medical School) were collected and used as a naturally secreted Aβ oligomers.
Aβ levels in the 3xTg-AD and 3xTg-AD/Fcgr2b KO mice were analyzed using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Briefly, the mice were anesthetized and the brains were microdissected. The hippocampus was carefully isolated and homogenized in 10 volumes of ice-cold guanidine buffer (5 M guanidine-HCl/50 mM Tris-Cl, pH 8.0) and then mixed for 3 hr at room temperature. The brain homogenates were further diluted 1:10 with cold reaction buffer (5% BSA, 0.03% Tween-20, and 5 mM EDTA in PBS supplemented with protease inhibitor cocktail) and then centrifuged at 16,000 g for 20 min at 4°C. The diluents were mixed 1:1 with standard dilution buffer in the assay kit.
Primary cortical and hippocampal neurons were cultured from embryonic day 17 (E17) mice. The neurons were plated on poly-L-lysine (0.01% in 100 mM borate buffer, pH 8.5)-coated glass coverslips and maintained in neurobasal medium containing 2% B-27 supplement (Invitrogen) and 0.5 mM L-glutamine (Invitrogen). Half of the medium was exchanged every 3 days. Primary neurons were not authenticated, and were not tested for mycoplasma. SH-SY5Y, HEK293T and CHO cells (ATCC, Manassas, VA), which were authenticated by ATCC and were negative for mycoplasma, were cultured in DMEM (HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS) (HyClone), penicillin and streptomycin (Invitrogen). Cells were grown at 37°C under an atmosphere of 5% CO2. Primary neurons were transfected using Lipofectamine 2000 reagent (Invitrogen), whereas other cells were transfected using Polyfect reagent (Qiagen, Germany) according to the manufacturer’s instructions. If required, cells were treated with SB-415286, roscovitine (Sigma-Aldrich) or AS1949490 (Tocris, United Kingdom) as indicated.
All primers used in this study are listed in Supplementary file 1. Human FCGR2B cDNAs was amplified by PCR from a human brain cDNA library and subcloned into pEGFP-N1 vector. The cDNAs of FCGR2B deletion mutants, ΔCyto, ΔITIM and ΔC-term was generated by PCR and subcloned into pEGFP-N1 vector. A point mutant of FCGR2B (Y273F) was generated by site-directed mutagenesis. All mutants were confirmed by DNA sequencing analysis. Human FCGR2B and human INPPL1 shRNAs were synthesized, annealed and cloned into the pSUPER-neo vector. Human tau (0N4R) cDNAs were subcloned into pcDNA3-HA and pEGFP-C1 vector as described previously (Park et al., 2012). His-tagged mouse Ship2 cDNA is a generous gift from Dr. M.G. Tomlinson (University of Birmingham, UK). GFP-tagged mouse Ship2 and D608A mutant were kindly provided by Dr. P. De Camilli (Yale University).
SH-SY5Y cell were transfected with pcDNA3-HA, pFCGR2B-HA, pSuper-neo, pFCGR2B shRNAs or pINPPL1 shRNAs for 36 hr and then cultivated in the selection medium containing 1 mg/ml G418 (Invitrogen) for at least two weeks. A single cell was further cultivated to form stable cell colony and the expression of each cell lines was analyzed by western blotting and reverse transcriptase (RT) PCR.
Cells were lyzed in lysis buffer (50 mM Tris-HCl pH 7.4, 30 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, 1 μg/ml each of aprotinin, leupeptin and pepstatin A). The lysates were centrifuged at 14,000 g for 10 min at 4°C and the supernatant was separated by SDS-PAGE and blotted onto PVDF membrane. The blots were blocked for 1 hr at room temperature and incubated with following antibodies: anti-phospho-FcγRIIb, anti-FcγRIIb (Epitomics, Burlingame, CA), anti-Aβ (4G8, Signet, Dedham, MA), anti-phospho-tau (AT180 and AT100, Innogenetics, Alpharetta, GA), anti-NSE (Zymed, South San Francisco, CA), anti-phospho-GSK3β, anti-p35/25 (Cell signaling, Danvers, MA), anti-GSK3β (BD Biosciences, San Jose, CA), anti-SHIP2, anti-mFcγRIIb, anti-GFP, anti-His (Santa Cruz Biotechnology Inc., Dallas, TX), anti-α-tubulin and anti-β-actin (Sigma-Aldrich), Nu-1 (kindly provided by Dr. W.L. Klein, Northwestern University), PHF1, CP13 and TG5 (a generous gifts from Dr. P. Davies, Albert Einstein College of Medicine), and 12E8 (a generous gift from Dr. P. Seubert (Elan Pharmaceuticals). Membranes were rinsed three times with TBS-T (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Tween-20), further incubated for 1 hr with peroxidase-conjugated secondary antibodies and visualized using ECL detection system.
Mouse primary cortical or hippocampal neurons were fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich) for 10 min, rinsed three times with PBS and then permeabilized with 0.1% Triton X-100 in PBS. After blocking with 5% BSA in PBS, neurons were incubated overnight at 4°C with the following antibodies: AT180 (1:200), AT8 (1:200), anti-NSE (1:200), anti-FcγRIIb (1:500) and anti-SHIP2 (1:250). After rinsing three times with PBS, cells were incubated with FITC- or TRITC-conjugated secondary antibodies (Jackson Laboratory Inc.) at room temperature for 1 hr. The coverslips were placed with mounting solution (Sigma-Aldrich) and observed on a confocal laser scanning microscope (Carl Zeiss Inc., Thornwood, NY).
For endogenous immunoprecipitation (IP) assay, Aβ1-42-treated SH-SY5Y cell extracts were incubated with anti-FcγRIIb or anti-SHIP2 antibodies in IP buffer [50 mM Tris-Cl (pH7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, a mixture of protease inhibitors] for 12 hr at 4°C, and then pulled-down by protein G-Sepharose beads (GE Healthcare, United Kingdom). For co-immunoprecipitation assay, HEK293T cells which transiently overexpressed His-tagged SHIP2 with either GFP-tagged FcγRIIb or FcγRIIb Y273F mutant were lyzed and incubated with anti-GFP or anti-His antibodies for 12 hr at 4°C, and then pulled-down by protein G-Sepharose beads. After a short centrifugation, the beads were washed three times with IP buffer and subjected to western blotting.
SH-SY5Y cells treated with Aβ1-42 were harvested with the buffer (20 mM Tris-HCl pH7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM EDTA) and then mechanically disrupted using a 26-gauge needle with passing it 20 times. Cell lysates were centrifuged at 1000 g for 10 min at 4°C to remove the nuclei or unbroken cells. The supernatant was collected into new tube and again centrifuged at 100,000 g for 1 hr at 4°C with Beckman SW41 rotor. The pellet was resuspended in lysis buffer and used as a crude membrane fraction, whereas the supernatant used as a cytosolic fraction. The separated fractions were confirmed with western blot analysis using anti-FcγRIIb and anti-α-tubulin antibodies for the membrane and cytosolic markers, respectively.
The lipid extraction from primary cortical neurons or SH-SY5Y cells was performed as described (Gray et al., 2003). After stimulation, cells were harvested with ice-cold 0.5 M trichloroacetic acid (TCA) solution, standing on ice for 5 min and centrifuged at 200 g for 5 min. The pellet was washed with 5% TCA with 1 mM EDTA solution. Neutral lipids were extracted from the pellet with a 2:1 solution of methanol and chloroform, followed by vigorous vortexing for 10 min at room temperature. The extracts were centrifuged at 200 g for 5 min, and the acidic lipids were then extracted. A 80:40:1 solution of methanol, chloroform and 12 M HCl was added to the pellet and vortexed for 15 min at room temperature, and then centrifuged at 200 g for 5 min. The 750 μl of supernatant was transferred to a new tube and added with 250 μl of chloroform and 450 μl of 0.1 M HCl. After mixing, the samples were centrifuged to separate the organic and aqueous phases and the lower organic phase was collected and dried under vacuum. The lipids were then resuspended by sonication in a water bath with an appropriate buffer (1:2:0.8 solution of chloroform, methanol and water for PLO assay, or PBS-T buffer for ELISA).
For the purification of recombinant proteins, the PH domain of TAPP1 and GRP1 was inserted into pET-28a vector. E.coli BL21 cells were transformed with the plasmids and cultured to reach an OD600 of 0.6, before induction with 1 mM IPTG. After incubation for 18 hr at 16°C, cells were harvested and lyzed by sonication. His-fused TAPP1-PH and GRP1-PH were purified from cell lysates using Ni-NTA chelating agarose CL-6B (Peptron, Korea). PLO assays were performed as described previously with minor modification (Dowler et al., 2002). Lyophilized PtdIns(3,4)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 diC16 (Echelon, Salt Lake City, UT) were reconstituted in a 1:2:0.8 solution of chloroform, methanol and water, and used as positive controls. The lipid extracts from the cells were serially diluted and spotted on PVDF membranes which were pre-wetted in methanol, washed in TBS-T buffer and then air-dried. The membranes were dried completely and blocked with 3% BSA in TBS-T buffer for 1 hr. The blots were incubated overnight at 4°C with gentle rocking in the fresh blocking buffer containing purified 10 μM His-TAPP1-PH or His-GRP1-PH. The membranes were washed 5 times over 50 min in TBS-T buffer and incubated with anti-His antibody for 1 hr at room temperature. The membranes were further incubated for 1 hr with peroxidase-conjugated secondary antibodies and bound proteins were visualized using ECL detection system. PtdIns(3,4)P2, PtdIns(4,5)P2 or PtdIns(3,4,5)P3 levels were quantified by ELISA kit (Echelon) following the manufacturer’s instructions.
The synthetic phosphoinositides diC16 were incubated with histone carriers (Echelon) with a 0.5–3:1 molar ratio for 15 min with a vigorous vortexing. The histone-phosphoinositides complex was diluted 1:10 with neurobasal media and added to primary cortical neurons. The phosphoinositides without carriers and the only carriers without phosphoinositides were used as negative controls.
Statistical analyses were performed with GraphPad Prism software. Differences between two means were assessed by paired or unpaired t-test. Differences among multiple means were assessed by one-way ANOVA, followed by Tukey’s post-hoc test. Error bars represent s.d. or s.e.m. as indicated.
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Bart De StrooperReviewing Editor; VIB Center for the Biology of Disease, KU Leuven, Belgium
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Thank you for submitting your article "FcγRIIb-SHIP2 axis links Aβ to tau pathology by disrupting phosphoinositide metabolism in Alzheimer's disease model" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor.
The work describes how FcγRIIb deficiency protects neurons against Aβ-induced toxicity. Especially the identification of SHIP2 linking Aβ to Tau pathology is novel and potentially of high interest. Overall the referees were in support of publication, but before reaching consensus a few critical questions should be addressed. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
1) Overall the paper should take more into account other publications in both Introduction and Discussion sections. There are in the main time at least ten other receptors for Aβ oligomers described. Also some critical perspective on what is meant with Aβ oligomers is indicated. For instance, the 7PA2 cell line also produces 'zeta' peptides and the relation between these in vitro generated mixtures of Aβ peptides and the in vivo situation is far from clear. It is important that the broader non-specialized reader of eLife is not misled in the sense that this paper resolves the controversies in the field. Therefore, critical literature should be included in the Introduction and Discussion, and some balancing of the conclusions in the current manuscript versus other work should be provided. Why do the authors think that their receptor is the major driver of pathology in AD and why are other receptors not considered? Does this need any qualifiers?
2) There are two major gaps in the proposed cascade and this should be mentioned at least in the Discussion to clarify this for the reader. First there are no mechanistic data in this work to link Aβ to FcγRIIb phosphorylation. The authors mention lyn kinase briefly (Discussion, third paragraph) but don't show any data. The second gap is a mechanistic link between increased PtdIns(3,4) and tau pathology. GSK-3-β kinase that catalyzes tau hyperphosphorylation, uses ATP as a donor of phosphate groups, so phosphoinositides are not a substrate. Therefore a possible link could be PtdIns(3,4)-mediated regulation of GSK-3β.
3) Two referees have concerns with the idea that the action of FcγRIIb is cell autonomous. First there are issues with the expression of FcγRIIb in neurons: Figure.1——figure supplement 1 A shows in fact that a large part of FcγRIIb mRNA is in glia and not in neurons. Additional evidence to proof that FcγRIIb is expressed in neurons is needed, it could be that the weak signal in the experiments in neurons is background. The authors could consider in situ hybridisation to check their claim. In addition, FcγRIIb is highly expressed in microglial cells and may play a role in microglial activation. Therefore, the impact of FcγRIIb KO on tau pathology in 3X TG mice can be also mediated by reduced microglial activation (cell autonomous effects vs non-autonomous effects). Did the authors check microglial activation and cytokine levels in these mice (WT 3XTG vs. KO 3Xtg)? Moreover, have the authors looked at whether FcγRIIb plays a role in sensing Aβ in microglial cells?
4) The authors should provide in the next version the pictures of Figure 1—figure supplements 2A-E which are now missing. These data are quite important as demonstrating the effect of the FcγRIIb receptor deficiency on APP processing.
5) In the last paragraph of the subsection “FcγRIIb is essential for tau hyperphosphorylation and memory deficit in 3xTg-AD mice”, it is said that 9months old 3xTgAD mouse are "plaque-free". What happens in the cortex of these 3xTg mice where Aβ plaques should be present, is tau phosphorylation more pronounced there? And is that protected by FcγRIIb deficiency? Does the age of mice affect tau hyperphoshorylation, i.e. is tau pathology still absent in 3xTg FcγRIIb KO mouse even at a very advanced age when there are loads of Aβ?
6) In Figure.3—figure supplement.2A the authors analyze the effects of the receptor mutations on Aβ-induced cell death. "Cell death was determined after 24h under fluorescence microscope". What method/dye/criterion was used? How many spots were analyzed per sample per condition to produce statistics on panel B?https://doi.org/10.7554/eLife.18691.029
[…] Essential revisions:
1) Overall the paper should take more into account other publications in both Introduction and Discussion sections. There are in the main time at least ten other receptors for Aβ oligomers described. Also some critical perspective on what is meant with Aβ oligomers is indicated. For instance, the 7PA2 cell line also produces 'zeta' peptides and the relation between these in vitro generated mixtures of Aβ peptides and the in vivo situation is far from clear. It is important that the broader non specialized reader of eLife is not misled in the sense that this paper resolves the controversies in the field. Therefore, critical literature should be included in the Introduction and Discussion, and some balancing of the conclusions in the current manuscript versus other work should be provided. Why do the authors think that their receptor is the major driver of pathology in AD and why are other receptors not considered? Does this need any qualifiers?
Until now, Aβ was reported to bind to many receptors, including alpha7 nicotinic acetylcholine receptors (α7 nAChR), NMDA receptor, receptors for advanced glycation end-products (RAGE), Aβ-binding alcohol dehydrogenase (ABAD), the Ephrin-type B2 receptor (EphB2), cellular prion protein (PrPc) and paired immunoglobulin-like receptor B (PirB) (Yan et al., 1996; Wang et al., 2000; Lustbader et al., 2004; Snyder et al., 2005; Lauren et al., 2009; Cisse et al., 2011a; Kim et al., 2013). Although these receptors were shown to be responsible for Aβ neurotoxicity, especially memory impairment in AD mice, their role as neuronal receptors in Aβ-induced tau pathologies was limitedly shown in α7 nAChR and NMDA receptor (reviewed in Stancu et al., 2014). Of particular note, while α7 nAChR was reported to mediate Aβ-induced tau phosphorylation, the finding was based on in vitroand ex vivosystem (Wang et al., 2003). It thus remains to be confirmed in detail using animal models of AD. Furthermore, evidence showing a correlation of the proposed molecular mechanism with pathologic evidence was not much provided. In particular, the CAMKK2-AMPK at down-stream of NMDA receptor was recently proposed to mediate the synaptotoxic effects of Aβ oligomers through tau phosphorylation and this event is very likely caused by NMDA receptor-induced increase of intracellular calcium, not by direct interaction of NMDA receptor with Aβ (Mairet-Coello et al., 2013). Therefore, a neuronal receptor that is important in Aβ- induced tau pathology has not been identified and needs to be elucidated for controlling overall AD pathology. We added this information in ‘Introduction’ (fourth paragraph).
Then, how is FcγRIIb different from other Aβ receptors? It is reasonable to propose that different mechanisms or even the same mechanism exerts multiple effects at different stages of disease progression (De Strooper and Karran, 2016). For instance, RAGE is now believed to mainly function to transport Aβ in the blood brain barrier (Deane et al., 2003) and ABAD acts for mitochondrial toxicity as an intracellular binding partner of Aβ (Lustbader et al., 2004). In case of PrPc, it's debatable whether it is involved in Aβ-induced memory impairments and thus needs to be further characterized (Balducci et al., 2010; Gimbel et al., 2010; Cisse et al., 2011b). Further, compared to those receptors, our observations that the phosphorylation of FcγRIIb at tyrosine 273 is found in the brain of AD patients and is required for both oligomeric Aβ neurotoxicity and tau hyperphosphorylation can make it distinct from other Aβ- binding receptors. In the case of PirB that shares structural similarity with FcγRIIb and also acts as an Aβ receptor for synaptic plasticity, the phosphorylation of PirB is not associated with Aβ signaling (Kim et al., 2013). Thus, we believe that FcγRIIb facilitates tau phosphorylation and neuronal loss in AD brains, consistent with the proposed role of tau in AD pathogenesis, such as severe memory impairment and neuronal loss (Ballatore et al., 2007). Following the reviewer’s suggestion, we added the specificity of FcγRIIb in ‘Discussion’ (fourth paragraph).
In general, Aβ oligomers play a key role in AD pathogenesis, while soluble Aβ oligomers are still heterogeneous, including low or high n oligomers, and the proposals on which species of Aβ oligomers are responsible for the pathogenesis are a little in debate (reviewed in Benilova et al., 2012). We have here used 3 different sources of Aβ oligomers; synthetic Aβ oligomers, naturally secreted Aβ oligomers (7PA2 cells), and Aβ of 3xTg-AD model mice. Although synthetic Aβ oligomers are well-characterized and have been used widely for neurotoxicity, the acting concentration of synthetic Aβ (μM range) is relatively higher than that in AD brains. Compared to synthetic Aβ oligomers, conditioned medium from 7PA2 cells mainly contains not only Aβ dimers and trimers but also the different pools of Aβ oligomers, including zeta peptide, and show more potent neurotoxic properties (nM range) (Qi-Takahara et al., 2005; Haass and Selkoe, 2007). Moreover, the oligomers generated in 3xTg-AD mouse brain may be more complicated and needs to be identified. Nonetheless, we propose here that FcγRIIb plays a crucial role in tau phosphorylation and neurotoxicity in vitroand in vivoin response to these species of Aβ, probably a certain common species among the different sources of Aβ oligomers. We added above information in ‘Discussion’ (second paragraph).
2) There are two major gaps in the proposed cascade and this should be mentioned at least in the Discussion to clarify this for the reader. First there are no mechanistic data in this work to link Aβ to FcγRIIb phosphorylation. The authors mention lyn kinase briefly (Discussion, third paragraph) but don't show any data. The second gap is a mechanistic link between increased PtdIns(3,4) and tau pathology. GSK-3-β kinase that catalyzes tau hyperphosphorylation, uses ATP as a donor of phosphate groups, so phosphoinositides are not a substrate. Therefore a possible link could be PtdIns(3,4)-mediated regulation of GSK-3β.
The first gap; Lyn is a member of Src-family tyrosine kinases and catalyzes the phosphorylation of FcγRIIb at ITIM domain for the inhibition of leukocyte signaling (Smith and Clatworthy, 2010). Thus, we initially hypothesized that and tested whether FcγRIIb was phosphorylated by Lyn after treatment with Aβ. We found that Lyn was activated (phosphorylation at Tyr 397) by Aβ1-42 treatment in primary cortical neurons and SH-SY5Y neuronal cells (Author response image 1A). In addition, we observed that knockdown of Lyn expression by shRNA suppressed Aβ- induced Lyn activation and FcγRIIb phosphorylation (tyrosine 273) (Author response image 1B). To further address the role of Lyn in AD, we also isolated a novel inhibitor of Lyn from virtual library. When we examined the activity of this Lyn inhibitor in vitroand in vivo, we consistently found that the Lyn inhibitor effectively prevented FcγRIIb-induced cell death (Author response image 1C) and Aβ-induced Lyn activation and FcγRIIb phosphorylation (Author response image 1D). Moreover, Lyn inhibitor reduced the Aβ-induced cell death in primary cortical neurons (Author response image 1E). Taken together, Lyn is required for FcγRIIb phosphorylation at Tyr273 for Aβ neurotoxicity. We have actually prepared these results together with other data of in vivotests using AD models as a separate issue, and are thus sorry to say that we would like to show these results only to reviewers. It also needs more detail characterization. Please, understand us. Thank you very much.
The second gap; the previous study showed that GSK3β, a well-known tau kinase, is stimulated by ER stress (Ren et al., 2015). We previously demonstrated that ER stress is elicited by the interaction between Aβ1-42 and FcγRIIb, and is responsible for Aβ neurotoxicity (Kam et al., 2013). Thus, we decided to examine the gap among SHIP2, ER stress, and GSK3β. To determine the relationship between PtdIns(3,4)P2 (a SHIP2 product) and ER stress, we first analyzed the alteration of GRP78, a typical marker of unfolded protein response (UPR), following the delivery of various phosphoinositides into primary cortical neurons. Among the phosphoinositides, we found that PtdIns(3,4)P2 only increased the level of GRP78. We also found that PtdIns(3,4)P2 abolished the inhibitory phosphorylation of GSK3β at Ser9 (GSK3β activation) and increased tau hyperphosphorylation (Figure 5—figure supplement 2A). In addition, we found that treatment with ER stress inhibitors, such as 4-PBA and Salubrinal, a chemical chaperone and an eIF2α dephosphorylation inhibitor, respectively, apparently attenuated PtdIns(3,4)P2-induced GSK3β activation and tau phosphorylation with some different efficacy (Figure 5—figure supplement 2B). The different efficacy of these two inhibitors on GSK3β and tau phosphorylation might reflect a way of their inhibition of ER stress and tau phosphorylation by kinases. These results suggest that GSK3β is activated by SHIP2 product through ER stress to phosphorylate tau. Further, treatment with AS1949490, a SHIP2 inhibitor, or infection with lentivirus carrying Ship2 shRNA blocked Aβ-induced GSK3β activation and tau hyperphosphorylation (Figure 5—figure supplement 2C, D), again confirming our proposal that activation of SHIP2 elicits ER stress to activate GSK3β and subsequent tau hyperphosphorylation. We included these results into supplementary information (Figure 5—figure supplement 2) (subsection “Dysregulation of phosphoinositide metabolism by the FcγRIIb-SHIP2 axis for tau 269 phosphorylation”, last paragraph).
3) Two referees have concerns with the idea that the action of FcγRIIb is cell autonomous. First there are issues with the expression of FcγRIIb in neurons: Figure 1—figure supplement 1 A shows in fact that a large part of FcγRIIb mRNA is in glia and not in neurons. Additional evidence to proof that FcγRIIb is expressed in neurons is needed, it could be that the weak signal in the experiments in neurons is background. The authors could consider in situ hybridisation to check their claim. In addition, FcγRIIb is highly expressed in microglial cells and may play a role in microglial activation. Therefore, the impact of FcγRIIb KO on tau pathology in 3X TG mice can be also mediated by reduced microglial activation (cell autonomous effects vs non-autonomous effects). Did the authors check microglial activation and cytokine levels in these mice (WT 3XTG vs. KO 3Xtg)? Moreover, have the authors looked at whether FcγRIIb plays a role in sensing Aβ in microglial cells?
To evaluate neuronal expression of FcγRIIb, we have really paid great attention and by performing many experiments.
First, as already shown in our previous manuscript (Kam et al., 2013), FcγRIIb mRNA (RT-PCR) and proteins (western blot analysis and immunostaining) were found in the neurons of mouse and AD patient brains (Author response image 2). Moreover, unlike other FcγRs, levels of FcγRIIb mRNA (RT-PCR) and proteins were greatly increased at least 3 to 10 folds by Aβ treatment in primary cultured neurons and NeuN-positive neurons in AD brains. For these analyses, we tested almost all of commercially available and home-made FcγRIIb antibodies (more than 7 antibodies) and consistently found similar results using a few of the antibodies.
Second, interestingly, we found a neuron-enriched isoform of FcγRIIb, FcγRIIb2, in the purified neurons and whole lysate of mouse hippocampus and cortex, whereas spleen and thymus express other isoform of FcγRIIb, FcγRIIb1 (Author response image 3A). Also, it is Fcgr2b2 mRNA that is selectively increased by oligomeric Aβ1-42 treatment in primary cortical neurons (Author response image 3B). From DNA sequencing analysis, we found that FcγRIIb2 is lacking one exon (IE1) in its cytoplasmic domain and exhibits a distinct function from FcγRIIb1, especially in neurotoxic activity. We are submitting a manuscript showing their functional differences in the neuropathologic activity in AD model to other journal and decided to show the results only to reviewer to support our current proposal.
Third, to evaluate neuronal activity of FcγRIIb, we know that generation of neuron- specific conditional knockout (nsKO) mice of FcγRIIb is the best. However, as you known well, generation of neuron-specific knockout (nsKO) mice of FcγRIIb and of FcγRIIb nsKO/3x AD double transgenic mice will take too long time (3 more years). Thus, as an alternative approach, we generated a dominant negative mutant of FcγRIIb (FcγRIIb DN; FcγRIIb-ΔCyto) lacking its cytoplasmic region (a.a. 232-300) and thus unable to transmit the signal into cells, and confirmed its protective activity in Aβ-induced neuronal cell death after its transient expression in neuronal cells (data not shown). We then generated a transgenic mouse expressing FcγRIIb- ΔCyto-HA under CamKIIα promoter, which drives neuron-specific expression of the transgene (Mayford et al., 1996) (Author response image 4A). We confirmed apparent but low expression of FcγRIIb-ΔCyto-HA in the hippocampus and cortex of the transgenic mice (Author response image 4B). To address neuropathic effect FcγRIIb in AD model mice, we next crossed the mice with 3xTg-AD model mice to generate 3xTg- AD/FcγRIIb-ΔCyto-HA double transgenic mice (Author response image 4C). The behavior tests of the mice showed that neuronal expression of FcγRIIb-ΔCyto-HA inhibited memory impairments of 3xTg-AD model mice in Y-maze, novel object recognition, and passive avoidance tests (Author response image 4D). These results are essentially similar with the results observed in 3xTg-AD/FcγRIIb KO double transgenic mice, and suggest that neuronal function of FcγRIIb is critical for the memory impairments in the AD model mice. As we mentioned above, these results together with those of neuron-enriched FcγRIIb2 isoform are included in other manuscript and are thus presented only here to provide more convincing evidence to reviewer.
Fourth, following the reviewer’s suggestion, we searched and found the neuronal expression of murine FcγRIIb by the in situhybridization (ISH) results provided from Allen Brain Atlas (http://www.brain-map.org/). Although fcgR2b expression is quite low, fcgr2b-positive ISH signals were detected in the hippocampus of P56 male mouse (Author response image 5).
Finally, consistent to our proposal, there are references showing the expression of FcγRIIb mRNA and proteins in neurons:
(1) Cahoy et al. [J. Neurosci. (2008)] created a transcriptome database of the expression levels of >20,000 genes by gene profiling from astrocytes, neurons and oligodendrocytes. Based on their list, FcγRIIb is significantly expressed in postnatal day 16 (P16) neurons and P17 forebrain. (2) Suemitsu et al. [Neuroscience (2010)] detected FcγRIIb protein in parvalbumin neurons, whereas FcγRIII and FcγRI proteins were detected in microglial cells.
In summary, we confirmed that, though neuronal expression is very low compared to non-neuronal cells, FcγRIIb is evidently expressed in neuronal cells as well. Again, please note that its expression is increased at least several folds in primary neurons by Aβ treatment and in AD brains compared to age-matched non- AD brains. Moreover, presence and distinct function of a neuron-enriched FcγRIIb2 provides more compelling evidence for the pathogenic role of FcγRIIb in AD.
While we have mainly focused on the role of neuronal FcγRIIb in tau pathology of AD model, we were also attempted to examine the role of FcγRIIb in the microglial activation in 3x Tg-AD mice. From immunohistochemical analysis, we identified that, compared to that of age-matched control mice, expression of Iba1, a microglia marker, was significantly reduced by Fcgr2b KO in the cortex (Author response image 6A, B) and slightly in the hippocampus (Author response image 6A, C) of 3x Tg-AD mice. In addition, RT-PCR analysis showed that mRNAs of pro-inflammatory genes (IL1β, iNOS, and TNFα) were also decreased by Fcgr2b KO in 3x Tg-AD mice (Author response image 6D). Though there is a tissue-specific difference, these observations suggest that ablation of FcγRIIb might reduce microglia activation in the brain of 3x Tg-AD mice. Unlike neuron-specific effects of FcγRIIb-ΔCyto-HA on tau phosphorylation and memory impairment seen in a transgenic mice expressing FcγRIIb-ΔCyto-HA, however, we do not think that the reduced inflammation by Fcgr2b KO in 3x Tg-AD mice is a direct cause of the memory rescue. At this moment, we do believe that the neuroinflammation via FcγRIIb also functions to contribute to the aggravation of memory impairment in AD mice, while we do not have evidence showing that the non-neuronal activity of FcγRIIb is solely responsible for the memory impairment. This role of FcγRIIb in the regulation of neuroinflammation is important and is worthwhile to pursue further in detail but is far away from our main story at this time. We showed these only to reviewers and discussed it in ‘Discussion’ as ‘data not shown’ (third paragraph).
4) The authors should provide in the next version the pictures of Figure 1—figure supplements 2A-E which are now missing. These data are quite important as demonstrating the effect of the FcγRIIb receptor deficiency on APP processing.
5) In the last paragraph of the subsection “FcJRIIb is essential for tau hyperphosphorylation and memory deficit in 3xTg-AD mice”, it is said that 9months old 3xTgAD mouse are "plaque-free". What happens in the cortex of these 3xTg mice where Aβ plaques should be present, is tau phosphorylation more pronounced there? And is that protected by FcγRIIb deficiency? Does the age of mice affect tau hyperphoshorylation, i.e. is tau pathology still absent in 3xTg FcγRIIb KO mouse even at a very advanced age when there are loads of Aβ?
In general, it is known that extracellular amyloid plaques were prominently detected over 12 month of age in 3x Tg-AD mouse model (Hirata-Fukae et al., 2008). Since we mostly analyzed tau phosphorylation at the age of 8-9 months, we performed additional experiments to identify the effect of FcγRIIb KO in more aged mice following the reviewer’s suggestion. While transgenic expression of human tau (detected by HT7 antibody) in 3x Tg-AD mice was not altered during aging, tau phosphorylation in 20 month-old 3x Tg-AD mice was heavily detected and occurred more than that of 6 month-old 3x Tg-AD mice (Figure 1-Figure supplement 3B, C). Compared to 15 month-old 3x Tg-AD mice, the level of hyperphosphorylated tau was reduced by Fcgr2b KO in the same age of 3x Tg-AD mice (Figure 1—figure supplement 3D, E). These results illustrate that tau phosphorylation mediated by FcγRIIb occurs at the onset of the disease and lasts to the late stage. We included these results (Figure 1—figure supplement 3) (subsection “FcγRIIb is essential for tau hyperphosphorylation and memory deficit in 3xTg-AD mice”, last paragraph).
6) In Figure 3—figure supplement 2A the authors analyze the effects of the receptor mutations on Aβ-induced cell death. "Cell death was determined after 24h under fluorescence microscope". What method/dye/criterion was used? How many spots were analyzed per sample per condition to produce statistics on panel B?
Apoptotic cell death was determined by morphology-based death assay as described in the previous literatures (Song et al., 2008; Han et al., 2010). After transfection with the DNAs, GFP-positive cells showing condensed and fragmented nuclei after staining with Hoechst 33258 were counted under a fluorescence microscope. We counted at least 900 cells in 3~4 random spots per sample per condition. We added this information into the figure legend of the revised manuscript (Figure 3—figure supplement 2A legend). To confirm this result of cell viability, we additionally determined their neurotoxicity using other cell viability assay in HT22 cells. We observed similar results as those mentioned above (Author response image 7).https://doi.org/10.7554/eLife.18691.030
- Tae-In Kam
- Hyejin Park
- Youngdae Gwon
- Seo-Hyun Kim
- Yong-Keun Jung
- Youngdae Gwon
- Sungmin Song
- Seo Won Moon
- Yong-Keun Jung
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
The authors thank Dr. U Hammerling (Memorial Sloan Kettering Cancer Center, NY) for FcγRIIb monoclonal antibody (K9.361 hybridoma), Dr. J Cambier (University of Colorado Health Sciences Center, CO) for FcγRIIb rabbit polyclonal antibody, Dr. DJ Selkoe (Harvard Medical School, MA) for CHO and 7PA2 cells, Dr. WL Klein (Northwestern University, IL) for oligomeric Aβ antibody, Dr. P Davies (Albert Einstein College of Medicine, NY) for PHF1, CP13, and TG5 antibodies, Dr. P Seubert (Elan Pharmaceuticals, CA) for 12E8 antibody, Dr. MG Tomlinson (University of Birmingham, Birmingham, UK) for SHIP1 and SHIP2 cDNAs, Dr. P De Camilli (Yale University, CT) for SHIP2 D608A cDNA. YD Gwon, SH Kim, and SW Moon were in part supported by the BK21 program and Global Ph.D. program. AD tissues were provided from the Harvard Brain Tissue Resource Center of McLean Hospital, MA. This work was support by the CRI grant (NRF-2016R1A2A1A05005304), the National Research Council of Science & Technology (NST) grant (MSIP Project No. 2N41660-16-074) and Global Research Laboratory (NRF-2010–00341) funded by the Ministry of Education, Science, and Technology.
Human subjects: All experiments using human brain samples were performed according to the protocol approved by the Seoul National University Institutional Review Board (SNU IRB, permit number: E1212/001-006).
Animal experimentation: All experiments involving animals were performed according to the protocols approved by the Seoul National University Institutional Animal Care and Use Committee (SNU IACUC) guidelines (Permit Number: SNU-130722-5-2).
- Bart De Strooper, VIB Center for the Biology of Disease, KU Leuven, Belgium
- Received: June 10, 2016
- Accepted: October 17, 2016
- Version of Record published: November 11, 2016 (version 1)
© 2016, Kam et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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